Lysozyme extraction from egg white using reverse micelles in a Graesser contactor: mass transfer characterization

The gentle mixing characteristics of a Graesser contactor can help to avoid the formation of stable emulsions, which is one advantage of this type of contactor when used with reversed micellar extraction. In this study, the performance of the Graesser contactor in lysozyme extraction from hen egg white is investigated. The concentration profile of lysozyme in the aqueous and organic phases indicated that, while substantial axial mixing occurred in the contactor, the extraction yield was in the range of 97% to 99%. The number of mass transfer units (N(ox)) was determined using a diffusion model, and the influence of aqueous-to-organic phase flow ratio, rotor speed, and total throughput on contactor performance was studied. It was found that the diffusion model could describe quite well the extraction of lysozyme from hen egg white using reversed micelles. The optimal conditions for the extraction at steady state were found to be a rotor speed of 5 rpm, an aqueous-to-organic phase flow ratio of 60:20 mL/min, and a total throughput of 80 mL/min. In addition, back-extraction was also performed using the conventional method (1.5 M KBr at pH 11.5) in the contactor. It was found that this mass transfer was not well described by a diffusion model, although 85% of the lysozyme could be recovered with the operating conditions used: a rotor speed of 10 rpm, and an aqueous-to-organic flow rate of 10:10 mL/min.     

Population-response model for vibrotactile spatial summation

A computational model based on previous physiological and psychophysical data is presented for the human Pacinian (P) psychophysical channel. The model can predict the probability of detection in simple psychophysical tasks, and hence psychometric functions and thresholds. The model simulates stimulating variable and fixed glabrous skin sites with different-sized contactors and includes spatial variation of monkey P-fiber sensitivities. Therefore, it is especially suitable for studying spatial summation, i.e. the improvement of threshold with increasing contactor area. Selective contributions of neural integration (n.i.) and probability summation (p.s.) are also incorporated into the model. Model predictions are compared to psychophysical results of Gescheider et al. (). The performance of the model regarding the effects of contactor size is very good. In addition to predicting approximately 3?dB improvement of thresholds when the contactor area is doubled, the model also reveals nonlinear contributions of p.s. and n.i. Furthermore, the model asserts that thresholds are largely governed by neural integration when small contactors are used. These and other findings discussed in the article show that the presented model is a helpful tool for formulating testable hypotheses. Although the model can also simulate some temporal summation effects, simulation results do not conform well to previous data on temporal response properties. Thus, the model needs to be refined in that respect.     

Population-response model for vibrotactile spatial summation

A computational model based on previous physiological and psychophysical data is presented for the human Pacinian (P) psychophysical channel. The model can predict the probability of detection in simple psychophysical tasks, and hence psychometric functions and thresholds. The model simulates stimulating variable and fixed glabrous skin sites with different-sized contactors and includes spatial variation of monkey P-fiber sensitivities. Therefore, it is especially suitable for studying spatial summation, i.e. the improvement of threshold with increasing contactor area. Selective contributions of neural integration (n.i.) and probability summation (p.s.) are also incorporated into the model. Model predictions are compared to psychophysical results of Gescheider et al. (2005). The performance of the model regarding the effects of contactor size is very good. In addition to predicting approximately 3 dB improvement of thresholds when the contactor area is doubled, the model also reveals nonlinear contributions of p.s. and n.i. Furthermore, the model asserts that thresholds are largely governed by neural integration when small contactors are used. These and other findings discussed in the article show that the presented model is a helpful tool for formulating testable hypotheses. Although the model can also simulate some temporal summation effects, simulation results do not conform well to previous data on temporal response properties. Thus, the model needs to be refined in that respect.     

Factors affecting tactile spatial acuity

Tactile spatial acuity on the fingerpad was measured using a grating orientation task. In this task, subjects are required to identify the orientation of square-wave gratings placed on the skin. Previous studies have shown that performance varies as a function of the width of the grooves in the gratings. In the present study, both groove width and the overall size and configuration of the contactors were varied. Sensitivity improved with wider grooves and with larger contactors. Additional measurements showed that the improved sensitivity is not the result of the increase in total area contacted, but rather is due to two other factors associated with larger contactors. One is the greater linear extent of the larger contactors. The other appears to be due to the reduction in the interference produced by the outer edge of the contactor. Specifically, as the contactor increases in size, the distance between the outer edge and the center portion of the grooves also increases. It was also shown that subjects are more sensitive to a single, continuous groove as compared with two grooves of the same total length but spatially discontinuous. Similarly, subjects are more sensitive to a contactor with a continuous groove than to a contactor in which just the end points of the groove are presented. The results are generally consistent with the results of peripheral, neurophysiological recordings. The results are discussed in terms of the way in which both spatial and intensive factors may affect sensitivity to grating orientation.     

Physical and biochemical characterization of a simple intermediate between fluidized and expanded bed contactors

Physical and biochemical comparison has been made of the performance of a simple fluidised bed contactor and a commercial expanded bed contactor, characterised by identical dimensions, and operated at various settled bed heights with two anion exchange adsorbents. The contactors were tested with various feedstocks comprising bovine albumin in the absence and presence of 20 g dry cell weight biomass litre^-1. Earlier classification of the simple contactor as a single-stage, well mixed fluidised bed is reviewed. The relative merits of STREAMLINE DEAE and DEAE Spherodex LS as fluidisable, anion exchange adsorbents are discussed.     

Multiplicity and stability analysis of microorganisms in continuous culture: effects of metabolic overflow and growth inhibition

Metabolic overflow (enhanced uptake of substrate and secretion of intermediates) is a phenomenon often observed for cells grown under substrate excess. Growth inhibition by substrate and/or product is also normally found for this kind of culture. An effort is made in this work to analyze the dynamic behavior of a continuous culture subject to metabolic overflow and growth inhibition by substrate and/or product. Analysis of a model system shows that in a certain range of operating conditions three nonwashout steady state solutions are possible. Local stability analysis indicates that only two of them are stable thus leading to multiplicity and hysteresis. Further analysis of the intrinsic effects of different terms describing the metabolic overflow and growth inhibitions reveals that for the model system and the parameters considered, the combined effects of product inhibition and an enhanced formation rate of product under substrate excess cause the multiplicity and hysteresis. Growth inhibition by substrate and/or an enhanced substrate uptake appear not to be necessary conditions. The combined effects of enhanced product formation and product inhibition can also lead to unusual dynamic behavior such as a prolonged time period to reach a steady state, oscillatory transition from one steady state to another, and sustained oscillations. Using the occurrence of multiplicity and oscillation as criteria, the operating regime of a continuous culture can be divided into four domains: one with multiplicity and oscillation, one with unique steady state but possible oscillatory behavior, the other two with unique and stable steady state. The model predictions are in accordance with recent experimental results. The results presented in this work may be used as guidelines for choosing proper operating conditions of similar culture systems to avoid undesired instability and multiplicity. Copyright 1998 John Wiley & Sons, Inc.     

Theory of the kinetics of reactions catalyzed by enzymes attached to membranes

A theoretical treatment has been worked out for the kinetics of solid-supported enzyme systems, with diffusive and electrostatic effects taken into account. A utilization factor, defined as the ratio of the actual reaction rate to the rate of substrate consumption in the outer solution, is defined, and equations to evaluate the utilization factor are given for five kinetic conditions: (a) Michaelis-Menten behavior, (b) substrate inhibition, (c) product inhibition (competitive), (d) product inhibition (noncompetitive), and (e) product inhibition (anticompetitive). When the solid-supported enzymes obey a Michaelis-Menten relationship, an equation for the apparent Michaelis constant is given and a criterion for insignificant diffusion effects is shown. A substrate-inhibited enzyme reaction may display multiple steady-state behavior, and a criterion for uniqueness is presented. In the case of product-inhibited enzyme reactions, the utilization factor is always less than that which corresponds to a Michaelis-Menten relationship. Equations to evaluate the apparent Michaelis and inhibition constants are given.     

Constant-ratio alternative substrate approach for differentiation of enzyme mechanisms

A new kinetic approach using alternative substrates as a tool for studying enzyme mechanisms is described. In this method the substrate to alternative substrate ratio is maintained constant and the common product (or summation of product analogs) is measured. The double-reciprocal plots so obtained at several constant ratios generate different patterns for various mechanisms, thus permitting a choice of kinetic model. In some cases, secondary intercept plots are utilized as a diagnostic aid. Another feature of this approach is that most of the resultant plots are linear. The graphical patterns for four cases of two-substrate, two-product reactions are presented as examples. These patterns allow one to differentiate several mechanisms which are not distinguishable by conventional alternative substrate, competitive inhibitor, or product inhibition studies alone. When used in combination with other methods, various mechanisms involving isomerization and abortive complex formation can be differentiated even if only one alternative substrate is available.     

Conventional and Dense Gas Techniques for the Production of Liposomes: A Review

The aim of this review paper is to compare the potential of various techniques developed for production of homogenous, stable liposomes. Traditional techniques, such as Bangham, detergent depletion, ether/ethanol injection, reverse-phase evaporation and emulsion methods, were compared with the recent advanced techniques developed for liposome formation. The major hurdles for scaling up the traditional methods are the consumption of large quantities of volatile organic solvent, the stability and homogeneity of the liposomal product, as well as the lengthy multiple steps involved. The new methods have been designed to alleviate the current issues for liposome formulation. Dense gas liposome techniques are still in their infancy, however they have remarkable advantages in reducing the use of organic solvents, providing fast, single-stage production and producing stable, uniform liposomes. Techniques such as the membrane contactor and heating methods are also promising as they eliminate the use of organic solvent, however high temperature is still required for processing.     

Kinetic resolution of chiral amines with omega-transaminase using an enzyme-membrane reactor

A kinetic resolution process for the production of chiral amines was developed using an enzyme-membrane reactor (EMR) and a hollow-fiber membrane contactor with (S)-specific omega-transaminases (omega-TA) from Vibrio fluvialis JS17 and Bacillus thuringiensis JS64. The substrate solution containing racemic amine and pyruvate was recirculated through the EMR and inhibitory ketone product was selectively extracted by the membrane contactor until enantiomeric excess of (R)-amine exceeded 95%. Using the reactor set-up with flat membrane reactor (10-mL working volume), kinetic resolutions of alpha-methylbenzylamine (alpha-MBA) and 1-aminotetralin (200 mM, 50 mL) were carried out. During the operation, concentration of ketone product, i.e., acetophenone or alpha-tetralone, in a substrate reservoir was maintained below 0.1 mM, suggesting efficient removal of the inhibitory ketone by the membrane contactor. After 47 and 32.5 h of operation using 5 U/mL of enzyme, 98.0 and 95.5% ee of (R)-alpha-MBA and (R)-1-aminotetralin were obtained at 49.5 and 48.8% of conversion, respectively. A hollow-fiber membrane reactor (39-mL working volume) was used for a preparative-scale kinetic resolution of 1-aminotetralin (200 mM, 1 L). After 133 h of operation, enantiomeric excess reached 95.6% and 14.3 g of (R)-1-aminotetralin was recovered (97.4% of yield). Mathematical modeling of the EMR process including the membrane contactor was performed to evaluate the effect of residence time. The simulation results suggest that residence time should be short to maintain the concentration of the ketone product in EMR sufficiently low so as to decrease conversion per cycle and, in turn, reduce the inhibition of the omega-TA activity.     

The effect of demulsifiers on lysozyme extraction from hen egg white using reverse micelles

The liquid–liquid extraction of protein from buffered aqueous phases using reverse micelles (RM) has been extensively researched from a fundamental point of view. However, very little effort has been expended at scaling up this process for the extraction of real fermentation broth. When real broths are used with reverse micellar phases there are major problems with emulsion formation. In this study the effect of a variety of demulsifiers on lysozyme extraction was evaluated in terms of their influence on the separating properties of the emulsion, water content (W _o ), and, extraction yield and kinetics from both buffer and hen egg white. In addition, the use of a low shear contactor (a Graesser or `raining bucket') was assessed in terms of its suitability as a RM contactor. It was found that most of the demulsifiers reduced the settling time of the emulsion, and enhanced the yield and kinetics of lysozyme extraction from hen egg white. It was hypothesised that this was due to the demulsifier displacing the lysozyme from the interface and preventing the protein unfolding and precipitating. This effect was found to depend on both the generic type of demulsifier, and its concentration.     

The hydrodynamics of a Graesser ("raining bucket") contactor with a reverse micellar phase

A variety of contactor types have been assessed for the liquid-liquid extraction of proteins using reversed micelles; however, many of these contactors suffer from drawbacks such as emulsion formation and poor mass transfer performance. In this study, a small (1.25 L) Graesser "raining bucket" contactor was assessed for use with this system since it has the potential to ameliorate many of these problems. The aim of the work was to evaluate the hydrodynamics of the contactor in order to use this information for future work on mass transfer performance. Hydrodynamic characteristics such as the axial mixing coefficient were determined by residence time distribution studies using a tracer injection method. The effect of rotor speed and flow rate of each phase on axial mixing was investigated, and as a result of its unusual structure, i.e., falling/rising sheet, the interfacial mass transfer area in the Graesser was determined by image analysis. It was found that rotor speed had more influence on the axial mixing coefficient in the aqueous phase than in the reverse micellar phase. The axial mixing coefficient in each phase increased by increasing the flow rate of the same phase. The images obtained in a dropping cell showed that under the conditions of this study (3 rpm, 22 degrees C), the bucket pours one phase through the other in the form of a curtain or sheet. A new image technique was developed to determine the interfacial area of both phases, and it was found that the specific area was 8.6 m(2)/m(3), which was higher than in a spray column but considerably lower than in a RDC or a Graesser run at high rotational speed (50 rpm) without the addition of a surfactant.     

Sources for structure formation and switches in metabolic pathways.

Zonation of function, i.e. localization of metabolic activity in certain regions of histologically uniform tissues, is an often observed phenomenon. Moreover, experiments show that such metabolic patterns are highly dynamical. Since in the pathways of intermediary metabolism no autocatalytic reactions are observed, different types of metabolic regulation are sources of the non-linearities necessary for structure formation. Two models of biochemical reactions frequently encountered in metabolic pathways, namely a bisubstrate kinetics model with substrate inhibition, and an allosteric model with product regulation, are presented. It is shown, that they are well-suited to reproduce the dynamical behavior suggested by experimental findings, like their capability to act as switches, or their ability for spatio-temporal pattern formation in mature tissues.     

Modeling product formation in anaerobic mixed culture fermentations

The anaerobic conversion of organic matter to fermentation products is an important biotechnological process. The prediction of the fermentation products is until now a complicated issue for mixed cultures. A modeling approach is presented here as an effort to develop a methodology for modeling fermentative mixed culture systems. To illustrate this methodology, a steady-state metabolic model was developed for prediction of product formation in mixed culture fermentations as a function of the environmental conditions. The model predicts product formation from glucose as a function of the hydrogen partial pressure (P(H2)), reactor pH, and substrate concentration. The model treats the mixed culture as a single virtual microorganism catalyzing the most common fermentative pathways, producing ethanol, acetate, propionate, butyrate, lactate, hydrogen, carbon dioxide, and biomass. The product spectrum is obtained by maximizing the biomass growth yield which is limited by catabolic energy production. The optimization is constrained by mass balances and thermodynamics of the bioreactions involved. Energetic implications of concentration gradients across the cytoplasmic membrane are considered and transport processes are associated with metabolic energy exchange to model the pH effect. Preliminary results confirmed qualitatively the anticipated behavior of the system at variable pH and P(H2) values. A shift from acetate to butyrate as main product when either P(H2) increases and/or pH decreases is predicted as well as ethanol formation at lower pH values. Future work aims at extension of the model and structural validation with experimental data.     

Static and dynamic behavior of a class of unstructured models of continuous bioreactors with growth associated product

The static and dynamic behavior of a class of unstructured models of continuous bioprocesses, for which the product is growth associated, are analyzed using elementary concepts of singularity theory and continuation techniques. The class consists of models for which both the rates of utilization of limiting substrate and product formation are linearly proportional to the specific cell growth rate. The kinetic expressions are allowed to assume general forms of substrate and nonbiomass product. The steady-state analysis allows the derivation of analytical results and the construction of a useful picture in the models' parameter space delineating the different static behavior these models can predict, including unique steady states and bistability. The analysis of the dynamic behavior allows the derivation of general analytical conditions for the occurrence of periodic behavior in the models. It is also shown that the subclass of these models for which the specific cell growth rate expression is monotonic with respect to the nonbiomass product is unable to predict a stable oscillatory behavior regardless of the expression of the growth rate. These results illustrate the fundamental weakness of this class of unstructured models in predicting transient behavior in continuous cultures. The effect of kinetic and operating parameters on the stability characteristics of these models is also investigated.     

Perspective on Water of Crystallization Affecting the Functionality of Pharmaceuticals

Water can be incorporated into crystalline lattice of organic molecules in several ways and thus forms systems with different molecular packing characteristics. This review outlines a general classification of hydrates and explains why it is of high relevance for pharmaceutical researchers to investigate water of crystallization and hydrate systems. The different manufacturing steps related to the final drug product are also briefly discussed with an emphasis on the role of water and possible solid-state transformations related to hydrates. Application of spectroscopic techniques in characterizing water of crystallization and the effect of water on the drug and formulation stability are presented. Moreover, the role of water on the in vitro drug dissolution behavior is reviewed where an example is shown to illustrate the recent development on correlating solid state of the drug, for instance, hydrate formation, to the dissolution profile of the product, to achieve a better understanding of the dissolution process.     

Substrate and energy costs of the production of exocellular enzymes by Bacillus licheniformis

Substrate and energy costs of the production of exocellular enzymes from glucose and citrate by B. Iicheniformis S1684 as well as molar growth yields corrected for these costs of product formation were calculated using data from chemostat experiments. The calculations showed that 1.46-1.73 mol glucose and 2.31-2.77 mol citrate are needed for formation and excretion of 1 mol protein. Consequently, the values of the maximal product yield from substrate (Y(psm') g/mol) are 80 < Y(psm) < 95 when product is formed from glucose and 50 < Y(psm) < 60 when product is formed from citrate. The higher substrate costs for product formation from citrate are due to a higher level of CO(2) production during protein formation and a higher substrate requirement for the energy supply of product formation and excretion than when product is formed from glucose. The theoretical ATP requirement for protein synthesis could be determined reasonably well, but the energy costs of protein excretion could not be determined exactly. The energy costs of protein formation are higher than those of biomass formation or protein excretion. Molar growth yields corrected for the substrate costs of product formation were high, indicating a high efficiency of growth.Growth and production parameters were determined as well from experimental data of recycling fermentor experiments using a parameter optimization procedure based on a mathematical model describing biomass growth as a linear function of the substrate consumption rate and the rate of product formation as a linear function of biomass growth rate. The fitting procedure yielded two growth and production domains during glucose limitation. In the first domain the values for the maximal growth yield and maintenance coefficient were in agreement with those found in chemostat experiments at corresponding values of Y(spm). Domain 2 could be described best with linear growth and product formation. In domain 2 the rate of product formation decreased and more substrate became available for biomass formation. As a consequence the specific growth rate increased in the shift from domain 1 to 2. Domain 2 behavior most probably is caused by the rel-status of B. Iicheniformis S1684.     

Mechanistic studies of Escherichia coli adenosine-5'-phosphosulfate kinase

Adenosine-5'-phosphosulfate kinase (APS kinase) catalyzes the formation of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the major form of activated sulfate in biological systems. The enzyme from Escherichia coli has complex kinetic behavior, including substrate inhibition by APS and formation of a phosphorylated enzyme (E-P) as a reaction intermediate. The presence of a phosphorylated enzyme potentially enables the steady-state kinetic mechanism to change from sequential to ping-pong as the APS concentration decreases. Kinetic and equilibrium binding measurements have been used to evaluate the proposed mechanism. Equilibrium binding studies show that APS, PAPS, ADP, and the ATP analog AMPPNP each bind at a single site per subunit; thus, substrates can bind in either order. When ATPgammaS replaces ATP as substrate the V(max) is reduced 535-fold, the kinetic mechanism is sequential at each APS concentration, and substrate inhibition is not observed. The results indicate that substrate inhibition arises from a kinetic phenomenon in which product formation from ATP binding to the E. APS complex is much slower than paths in which product formation results from APS binding either to the E. ATP complex or to E-P. APS kinase requires divalent cations such as Mg(2+) or Mn(2+) for activity. APS kinase binds one Mn(2+) ion per subunit in the absence of substrates, consistent with the requirement for a divalent cation in the phosphorylation of APS by E-P. The affinity for Mn(2+) increases 23-fold when the enzyme is phosphorylated. Two Mn(2+) ions bind per subunit when both APS and the ATP analog AMPPNP are present, indicating a potential dual metal ion catalytic mechanism.     

A general solution for the steady-state kinetics of immobilized enzyme systems

A power series solution is presented which describes the steady-state concentration profiles for substrate and product molecules in immobilized enzyme systems. Diffusional effects and product inhibition are incorporated into this model. The kinetic consequences of diffusion limitation and product inhibition for immobilized enzymes are discussed and are compared to kinetic behavior characteristic of other types of effects, such as substrate inhibition and substrate activation.     

Characterization of a new gas-liquid contactor for the biological treatment of gaseous industrial effluents

This paper deals with a new type of gas-liquid contactor for the biological treatment of volatile organic compounds (VOCs). The hydrodynamic and the mass transfer characteristics of this contactor are studied. With head losses quite adapted to the use of blowing engines, the mass transfer capacity is found to be much higher than the mass flowrates which are normally contained in the industrial effluents.